A Serratia marcescens SM2306 strain for degrading dimples, and preparation method and application thereof

Abstract

The invention discloses a Serratia marcescens SM2306 strain for degrading dimples, a preparation method and an application thereof. The strain is Serratia marcescens, which is classified and named as Serratia marcescens ( Serratia marcescens ); deposited in the General Microbiological Center of China Microbiological Culture Collection Committee, the deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing; deposit date: March 25, 2024; deposit number: CGMCC No. 30142. The Serratia marcescens SM2306 of the present invention is a degrading bacterium that can be used to degrade toxic dibu of Solanaceae plants, has the advantages of strong degradation ability and environmental safety, and can be used for biodegradation of dibu.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, in particular to a Serratia marcescens SM2306 strain capable of degrading dimples, and a preparation method and application thereof. Background Art

[0002] DIMBOA (2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one), also known as hydroxamic acid, is a benzoxazinone compound. It is a toxic and secondary metabolite in corn and other gramineous crops. It is still toxic to corn borers and armyworms, and plays an important role in the defense of corn and other gramineous crops against pests. DIMBOA substances are mainly stored in the form of glycosylation. They exert their insect resistance by losing the glycosylation in the body of the pest and converting into toxic ligands such as DIMBOA and HDMBOA. They are key indicator compounds for pest control.

[0003] Converting toxic dimbu into non-toxic substances is one of the research topics at home and abroad. The degradation and metabolism of dimbu is usually transformed into dimbu and other chemical substances under the action of degrading enzymes. Some scholars have also isolated bacteria that can degrade and metabolize dimbu from the soil, but the degradation efficiency is low. In addition, the isolation rate of dimbu-degrading bacteria obtained by traditional separation and purification from soil is low. Therefore, seeking microorganisms that can efficiently degrade dimbu is an important way to quickly and safely degrade dimbu.

[0004] Serratia marcescens, also known as Bacillus prodigiosus, is a bacterium that produces bright red pigment. It was first discovered by an Italian pharmacist in 1819 and named in 1823. It was classified as Halomonas at the time and named Monas prodigiosus. Later, it was classified as Bacillus and named Bacillus prodigiosus. Many scholars have conducted biological tests on the biocontrol potential of Serratia marcescens strains against pests, such as the control of Asian citrus psyllid Diaphorina citri (Kuwayama) nymphs, yellow-legged locust Oedaleus infernalis Saussure, brown planthopper Nilaparvata lugens (Stal), and beet armyworm Spodoptera exigua (Hubner) by different Serratia marcescens strains.

[0005] The fall armyworm Spodoptera frugiperda, belonging to the genus Spodoptera of the family Noctuidae in the order Lepidoptera, has been causing damage in my country's corn-growing areas since it was discovered in Jiangcheng County, Yunnan Province on January 11, 2019. The fall armyworm larvae contain abundant symbiotic bacteria in their intestines, which play an important role in the adaptation of the potato tuber moth host, as well as the digestion and metabolism of nutrients. The degradation of insect-resistant substances in host plants by symbiotic bacteria in the insect intestine has attracted attention at home and abroad.

[0006] At present, there is a lack of a Serratia marcescens SM2306 strain for degrading diatomaceous earthwood with strong degradation ability and environmental safety and application thereof. Summary of the invention

[0007] In view of the above-mentioned defects of the prior art, the present invention provides a Serratia marcescens SM2306 strain for degrading taffeta with strong degradation ability and environmental safety and application thereof.

[0008] In order to solve the problems of the prior art, the present invention provides the following technical solutions: In a first aspect, the present application provides a Serratia marcescens SM2306 strain for degrading DIMB.

[0009] In a second aspect, the present application provides a Serratia marcescens bacterial agent prepared by the Serratia marcescens SM2306 strain for degrading DIMB.

[0010] In a third aspect, the present application provides a method for preparing a Serratia marcescens agent.

[0011] In a fourth aspect, the present application provides a use of a Serratia marcescens SM2306 strain or a Serratia marcescens agent in the preparation of a DIMBAL degradation preparation.

[0012] The first aspect of the present application provides a Serratia marcescens SM2306 strain for degrading dincloth. The strain is Serratia marcescens, which is classified and named Serratia marcescens ( Serratia marcescens ); deposited in the General Microbiology Center of China Microbiological Culture Collection Administration, the deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing; deposit date: March 25, 2024; deposit number: CGMCC No. 30142.

[0013] Furthermore, the nucleotide sequence of 16S rDNA of Serratia marcescens SM2306 strain is shown in SEQ ID NO.1.

[0014] Furthermore, Serratia marcescens SM2306 is a Gram-negative bacterium with a short rod-shaped cell morphology and a size of 1-1.3 μm×0.7-1.0 μm. After streak purification and culture on an LB plate, the SM2306 colonies were round with neat edges, orange-yellow color, smooth and moist surface, slightly convex in the center, and flat around.

[0015] The second aspect of the present application provides a Serratia marcescens bacterial agent prepared by the Serratia marcescens SM2306 strain for degrading dincloth.

[0016] Furthermore, the active ingredient is at least one of the following (a), (b), and (c):

[0017] (a) Primary extract of fermentation broth of Serratia marcescens SM2306 strain;

[0018] (b) the supernatant of ultrasonic lysis of Serratia marcescens SM2306 strain cells;

[0019] (c) The obtained ultrasonic lysis precipitate of Serratia marcescens SM2306 strain cells.

[0020] The third aspect of the present application provides a method for preparing a Serratia marcescens inoculum, comprising the following steps:

[0021] (1) The Serratia marcescens SM2306 strain was isolated from the midgut of the fall armyworm larvae and cultured in LB medium;

[0022] (2) Purifying on LB medium to obtain a purified Serratia marcescens inoculum;

[0023] (3) The degradation capacity of DIBU was measured using a bacterial solution of a high-efficiency degradation strain, and the degradation rate of DIBU reached 90%.

[0024] The fourth aspect of the present application provides a use of a Serratia marcescens SM2306 strain or a Serratia marcescens agent in the preparation of a diatomaceous earth degradation preparation.

[0025] Beneficial effects: The Serratia marcescens SM2306 provided by the present invention is a degrading bacterium that can be used to degrade toxic dibothia serrata of Solanaceae plants, has the advantages of strong degradation ability and environmental safety, and can be used for biodegradation of dibothia serrata.

[0026] Compared with the prior art, the present invention has the following advantages: (1) Highly efficient degradation ability: The Serratia marcescens SM2306 strain provided by the present invention has a strong degradation effect on dimpling, and its degradation efficiency is as high as 90%, which can effectively convert toxic dimpling into non-toxic substances. Easy to culture and reproduce: The Serratia marcescens SM2306 strain is simple to culture, grows quickly, and is easy to culture and reproduce in large quantities indoors, which is conducive to industrial production and application.

[0027] (2) Environmental safety: The strain will not produce any substances harmful to the environment during the degradation of tinfoil, and is environmentally friendly. It is a green and safe biodegradation method. Food safety assurance: By using the Serratia marcescens SM2306 strain to degrade tinfoil, the safety of food developed with tinfoil-containing materials as raw materials can be guaranteed, reducing the potential risks of tinfoil to human health.

[0028] (3) Broad application prospects: This strain can not only improve the adaptability of pests such as fall armyworm to gramineous plants rich in dimb, but also efficiently degrade dimb. It has broad application prospects and can be widely used in the biodegradation of dimb, effectively reducing environmental pollution caused by chemical degradation. Improve pest adaptability: As an insect intestinal symbiotic bacteria, this strain can improve the adaptability of pests such as fall armyworm to gramineous plants rich in dimb, which is helpful for the biological control of agricultural pests.

[0029] Promoting sustainable agricultural development: Through biodegradable dingbu, the use of chemical pesticides can be reduced, promoting the sustainable development of agriculture, while reducing the impact of agricultural production on the environment. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 It is a cell morphology diagram of Serratia marcescens SM2306 of the present invention.

[0031] Figure 2 It is a growth curve diagram of Serratia marcescens SM2306 of the present invention. DETAILED DESCRIPTION

[0032] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present application more clearly understood, the present application is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application.

[0033] In a first aspect, the present invention provides a Serratia marcescens SM2306 strain for degrading dimples. The strain is Serratia marcescens, which is classified and named Serratia marcescens ( Serratia marcescens ); deposited in the General Microbiology Center of China Microbiological Culture Collection Administration, the deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing; deposit date: March 25, 2024; deposit number: CGMCC No. 30142.

[0034] In some embodiments, the nucleotide sequence of 16S rDNA of Serratia marcescens SM2306 strain is shown as SEQ ID NO.1.

[0035] In some embodiments, the Serratia marcescens SM2306 strain is a Gram-negative bacterium with a short rod-shaped cell morphology and a size of 1-1.3 μm×0.7-1.0 μm. After streak purification and culture on an LB plate, the SM2306 strain has a round colony with neat edges, an orange-yellow color, a smooth and moist surface, a slightly convex center, and flat surroundings.

[0036] A second aspect of the embodiments of the present application provides a Serratia marcescens bacterial agent prepared by the Serratia marcescens SM2306 strain for degrading DIMB.

[0037] In some embodiments, the active ingredient is the primary extract of the fermentation broth of Serratia marcescens SM2306 strain;

[0038] In some embodiments, the active ingredient is the supernatant of ultrasonic lysis of Serratia marcescens SM2306 strain cells;

[0039] In some embodiments, the active ingredient is the ultrasonic lysis precipitate of Serratia marcescens SM2306 strain cells.

[0040] The third aspect of the embodiment of the present application provides a method for preparing a Serratia marcescens inoculum agent, comprising the following steps:

[0041] (1) The Serratia marcescens SM2306 strain was isolated from the midgut of the fall armyworm larvae and cultured in LB medium;

[0042] (2) Purifying on LB medium to obtain a purified Serratia marcescens inoculum;

[0043] (3) The degradation capacity of DIBU was measured using a bacterial solution of a high-efficiency degradation strain, and the degradation rate of DIBU reached 90%.

[0044] A fourth aspect of the embodiments of the present application provides a use of a Serratia marcescens SM2306 strain or a Serratia marcescens agent in the preparation of a DIMBAL degradation preparation.

[0045] Example 1

[0046] The invention discloses a Serratia marcescens SM2306 strain for degrading diaper. The strain is Serratia marcescens and is classified and named as Serratia marcescens. The strain is deposited in the General Microbiological Center of China National Committee for the Preservation of Microorganisms, and the deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is March 25, 2024. The deposit number is CGMCC No. 30142.

[0047] The nucleotide sequence of 16S rDNA of Serratia marcescens SM2306 strain is shown in SEQ ID NO.1.

[0048] Serratia marcescens SM2306 is a Gram-negative bacterium with a short rod-shaped cell morphology and a size of 1-1.3 μm×0.7-1.0 μm. After streak purification and culture on an LB plate, the SM2306 colony is round with neat edges, orange-yellow color, smooth and moist surface, slightly convex in the center, and flat around.

[0049] The present invention discloses a Serratia marcescens bacterial agent prepared by the Serratia marcescens SM2306 strain for degrading dincloth. The active ingredient thereof is at least one of the following (a), (b) and (c):

[0050] (a) Primary extract of fermentation broth of Serratia marcescens SM2306 strain;

[0051] (b) the supernatant of ultrasonic lysis of Serratia marcescens SM2306 strain cells;

[0052] (c) The obtained ultrasonic lysis precipitate of Serratia marcescens SM2306 strain cells.

[0053] A method for preparing a Serratia marcescens inoculant of the present invention comprises the following steps:

[0054] (1) The Serratia marcescens SM2306 strain was isolated from the midgut of the fall armyworm larvae and cultured in LB medium;

[0055] (2) Purifying on LB medium to obtain a purified Serratia marcescens inoculum;

[0056] (3) The degradation capacity of DIBU was measured using a bacterial solution of a high-efficiency degradation strain, and the degradation rate of DIBU reached 90%.

[0057] The invention discloses an application of a Serratia marcescens SM2306 strain or a Serratia marcescens bacterial agent in preparing a diatomaceous earth degradation preparation.

[0058] The inventors' previous studies have found that Serratia marcescens is an important endosymbiotic intestinal bacterium in the intestine of fall armyworm, with an abundance of more than 40% in the intestine of fall armyworm larvae. It has a high efficiency in degrading dinkum, with a degradation efficiency of up to 86.5%. Therefore, the use of Serratia marcescens SM2306 strain isolated from the midgut of fall armyworm larvae to degrade dinkum has important practical application value for the degradation of dinkum in foods developed with dinkum-containing materials as raw materials, ensuring food safety, and providing selectable strains.

[0059] Test Example 1

[0060] Isolation and identification of Serratia marcescens SM2306

[0061] 1.1 Materials

[0062] 1.1.1 Test strains

[0063] The strain was isolated on June 20, 2023 from the midgut of the third instar larvae of Spodoptera frugiperda in the corn field in Wangtouzhai Village, Pupiao Town, Longyang District, Baoshan City, Yunnan Province (N 25°0'17", E 99°0' 21", 1370 m above sea level).

[0064] 1.1.2 Isolation and culture of strains

[0065] Take 50 healthy third-instar larvae of fall armyworm of the same size, repeat 3 times, a total of 150, and starve them in a sterile culture dish for 8 hours. Clean the clean bench surface with 75% alcohol first, disinfect all dissecting instruments with 75% alcohol, and then irradiate with ultraviolet light for more than 30 minutes to complete the disinfection of the clean bench. Then freeze the starved fall armyworm larvae at -20℃ for 2-3 minutes, wait for them to be paralyzed, rinse them with sterile water twice, 1 minute each time, disinfect them with 75% alcohol for 90 seconds, and finally rinse them with sterile water twice, 1 minute each time. After taking them out, place them in a sterile and clean culture dish, remove the midgut from the tail of the insect body with sterile tweezers, dissolve the contents in a centrifuge tube containing 1 mL of sterile PBS buffer, and fully homogenize. The purified strain is stored in a 4℃ refrigerator using LB slant medium.

[0066] 1.1.3 Species identification

[0067] Morphological identification: Pick a single colony of the highly efficient degradation strain obtained by the above screening and streak it on LB medium, culture it at 25℃ for 72 hours, observe and record the morphology, color, ridge, edge shape, dryness and wetness of the single colony. Stain the strain with Gram staining to observe the cell morphology and color. Purple represents positive bacteria and red represents negative bacteria. Refer to the methods of the "Common Bacterial System Identification Manual" and the "Berger Bacterial Identification Manual" for identification.

[0068] Molecular identification:

[0069] First, the bacterial DNA was extracted by freeze-thaw method. The specific operation steps are as follows: the strains were inoculated into LB liquid culture medium, placed in a shaker at 25°C and 180 r / min for 48 h. Take 2 mL of bacterial solution and place it in a centrifuge tube, centrifuge it at 12000 r / min for 2 min, and discard the supernatant. Add 500 μL of sterile water to the above precipitate, vortex and oscillate for 1 min to completely suspend and mix the bacteria. Then freeze with liquid nitrogen for 10 min, boil in water bath for 5 min, centrifuge at 12000 r / min for 2 min, and take the supernatant as PCR template DNA for later use.

[0070] Secondly, the 16S rRNA gene was amplified using bacterial universal primers 27f and 1492r. The reaction system was 25 μL: 1 μL of each primer, 1 μL of template DNA, 12.5 μL of 2ÍTaq PCR master mix, and 9.5 μL of dd H2O. Reaction conditions: 94℃ heat denaturation for 5 min; 94℃ denaturation for 1 min, 53℃ annealing for 1 min, 72℃ extension for 2 min, 32 cycles, and 72℃ extension for 10 min. Take 3 μL of PCR product for gel electrophoresis detection. Thirdly, the identified PCR product was sent to Sangon Biotech (Kunming) Co., Ltd. for sequencing. Fourthly, the sequencing results were spliced ​​using Contigexpress software, and the spliced ​​16SrDNA sequence was compared with the data in the NCBI database through the NCBI Blast program to find the most similar typical strain and download the sequence (as shown below). Fifth, MEGA7 software was used to construct a phylogenetic tree using the internationally accepted neighbor-joining method and Kimura two-parameter correction model. Bootstrap validation was performed with 1000 repeated samplings to analyze and evaluate the stability of the topological structure of the phylogenetic tree.

[0071] 16S sequence number of Serratia marcescens SM2306 strain:

[0072]

[0073] 1.2 Results

[0074] The LB plate streak purification culture showed that the colonies of strain SM2306 were round, with neat edges, light yellow, slightly transparent, moist, and low convex surface. Gram staining showed red, Gram-negative bacteria, and the cell morphology was rod-shaped ( Figure 1 ).

[0075] The growth curve results of the culture of the Serratia marcescens SM2306 strain showed that there was no lag phase, 0-6 h was the logarithmic growth phase, with the fastest growth at 2-4 h; 6-28 h was the stable phase, with the maximum growth reaching 24 h and then slowly declining; 28-48 h was the decay phase ( Figure 2 is the growth curve of Serratia marcescens SM2306 of the present invention).

[0076] Test Example 2

[0077] Degradation of DIMB by Serratia marcescens SM2306

[0078] 1 Materials

[0079] The Serratia marcescens SM2306 strain was used as the material. 5 mL of a single colony of Serratia marcescens was inoculated into the LB liquid medium prepared in advance and cultured at 25°C and 180 rpm with shaking until the OD 600 nm =1.0.

[0080] Induction: Take 1 mL of Serratia marcescens culture solution in a 100 mL Erlenmeyer flask and add it to 9 mL of LB medium to make the final OD 600 nm = 0.1; supplement with Serratia marcescens buffer (control) or 5 µg / mL DIB, and culture at 180 rpm and 25°C for 20 h. Centrifuge the Serratia marcescens bacterial suspension at 4000×g for 20 min at 25°C, and the supernatant is the crude extract of Serratia marcescens exoenzyme. Repeat this process 3 times.

[0081] Extracellular enzyme degradation system: Take 100 µL of 50 mM sodium acetate buffer at pH 5.5, 400 µL of 100 mM sodium acetate buffer at pH 5.0 (0.2 mM dABA) substrate, and 450 µL of extracellular crude extract, a total of 950 µL, and incubate at 25°C for 35 h. Heat at 90°C for 10 min to terminate the enzyme reaction, then centrifuge at 17,000 × g for 10 min and transfer the supernatant. Use it for LC-MS analysis of intermediate metabolites produced during the degradation process.

[0082] 2. Results

[0083] The degradation ability of DINB was determined by using the extracellular enzyme of the efficient degradation strain. The results showed that the degradation rate of Serratia marcescens SM2306 strain was 90%. According to the determination of the degradation ability of Serratia marcescens SM2306 strain extracellular enzyme on DINB, the degradation rate of Serratia marcescens SM2306 strain extracellular enzyme on DINB was 90%. This shows that Serratia marcescens SM2306 strain has a strong degradation ability on DINB.

[0084] In summary, the Serratia marcescens SM2306 strain of the present invention is an insect intestinal symbiotic bacterium that can be used to degrade diaper, has the advantages of strong degradation effect and no environmental pollution, and can be used in the practice of biodegradation of diaper.

[0085] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments, and the above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, and the scope of protection of the present invention is defined by the attached claims, description and their equivalents.

Claims

1. A Serratia marcescens that degrades dinclot ( Serratia marcescens) strain SM2306, Features: The strain is deposited in the General Microbiology Center of China Microbiological Culture Collection Administration, with the deposit address at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; deposit date: March 25, 2024; deposit number: CGMCC No. 30142.

2. The Serratia marcescens bacterial agent prepared by the Serratia marcescens SM2306 strain for degrading dincloth according to claim 1.

3. The method for preparing the Serratia marcescens inoculant of claim 2, characterized in that The steps include: (1) The Serratia marcescens SM2306 strain of claim 1 is cultured in LB medium; (2) The bacterial strain is then purified on LB medium to obtain a purified Serratia marcescens inoculum.

4. Use of the Serratia marcescens SM2306 strain according to claim 1 or the Serratia marcescens inoculant according to claim 2 in the preparation of a DIMBAL degradation preparation.

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